We are studying key cellular processes related to the tropic action of ACTH and LH on steroidogenesis in rat adrenocortical carcinoma cells and rat Leydig tumor cells, respectively. We have determined that cholesterol synthesized de novo as well as high-density and low-density lipoprotein cholesterol under appropriate conditions may all be major sources of the cholesterol utilized for steroidogenesis in these tumor cells. Cells in primary monolayer culture appear to be more dependent on lipoprotein cholesterol for steroidogenesis than freshly dispersed cells from solid tumors. The tumor cells have a limited capacity to metabolize cholesterol esters and, thus, the regulation of the mobilization of nonesterified cholesterol within the cells is an important determinant of the rate of steroid production. The participation of sterol-binding proteins as well as processes involving calcium ions are under active investigation. We also are studying the chronic tropic hormonal regulation of the synthesis of components of the cholesterol side-chain cleavage enzyme, the rate-limiting step of steroidogenesis. After cell proteins are radiolabeled with [35S]methionine, the enzyme components are immunoisolated specifically, using IgG fractionsof antisera to bovine adrenal cytochrome P-450scc, adrenodoxin and adrenodoxin reductase. We shall clarify whether ACTH or LH have a chronic effect on tumor cell steroidogenesis by stimulating enzyme synthesis. We are determining, using a similar approach, if serum or growth factors may serve as putative second tropic agents for adrenal and Leydig tumor cells by promotion of cell growth and synthesis of steroidogenic enzymes and proteins.